METHYLENE BLUE REDUCTION TEST (MBRT) To Check The Quality of Milk In a Microbiology Lab

In the methylene blue reduction (MRBT) test 1 ml of methylene blue (1: 25,000) is added to 10 ml of milk. The tube is sealed with rubber stopper and slowly inverted three times to mix. It is placed in a water bath at 35°C and examined at intervals up to 6 h. The time it takes for the methylene blue to become colourless is the methylene blue reduction time (MBRT). The shorter the MBRT, the lower the quality of milk.

Table: Grading of milk samples on the basis of methylene-blue reduction test in    different milk samples.

Quality of milk                                 Decolourization time

    Excellent                                        More than 5 h 

     Good                                              Between 4 hours and 5 h

      Fair                                               Between 3 to 4 h

      Poor                                               Less than 2 h

Effects of Genetic Manipulation

Genetic manipulation is an un-natural practice by which the scientists can produce any organism with desired traits. Along with some advantages, this manipulation can have several harmful effects over the society which are discussed as follows:

1.    1. An organism which is born through un-natural means can have an untested genetic build up and if such genes become a part of the society can lead to many disasters.

2.  2.  It can also cause an ecological imbalance. The new organism can be resistant to certain drugs and antibiotics which can lead to the introduction of certain resistant variety of a disease causing harmful microbe.

3.   3. There are high chances that the information of changing the genetic make up could be misused.

4.    4. The genetically modified foods can cause an allergy among certain people who are allergic to a particular chemical. Moreover they will become the part of out genetic structure and can cause mutations at small level in our DNA.

5.  5.   The organisms which are used for testing die an un-natural death or have to undergo a lot of pain so they are given “mercy killing” in the end (As in case of Sheep clone Dolly). This practice of torturing the animals is unacceptable.

6.    6. There is also a risk of generating a superior human species which can be misused. 

7.   7.  There is a small but significant risk of the transmission of usually fatal zoonotic diseases. The introduction of these diseases to the human population could have devastating consequences.

8.  8.   There are other unknown risks related to environment. 

9.  9.  The genetically modified crops will prove expensive to the farmers because the seeds of such crops cannot be used to sow the crop again. So the farmer has to buy the seed every time. (As in case of BT cotton)

1. One of the major factor of concern is Bioterrorism where a deadly pathogen is used as a weapon. Bioterrorism can lead to huge disasters and loss of several lives across the globe.

WESTERN BLOT

A Western Blot (immunoblotting)is an analytical technique used for detection of specific proteins. The protein from the sample is first separated according to size by SDS-PAGE (Polyacrylamide Gel Electrophoresis) and then transferred to a nitrocellulose or Polyvinylidene Fluoride (PVDF) membrane where they become immobilized. Then they are detected first by a specific primary antibody and then by enzyme linked secondary antibody. The enzyme linked to the secondary antibody forms a colored band after conjugation which shows the presence of specific protein. It is used as a confirmatory test in many medical diagnostic tests like HIV, because it gives information if a specific antibody is been secreted against the pathogen.

Alpha Complementation method for Bacterial Screening

Alpha complementation is a method for bacterial screening that have been transformed with a plasmid vector, carrying the N-terminal coding sequence for β-galactosidase of the lac operon. The lac Z gene is required for galactosidase metabolism. The pUC plasmid carries the alpha fragment of the lac Z gene which is the amino-terminus of the protein and is non functional alone. The omega fragment is found in E. coli chromosome, which is the carboxyterminus of the protein and  is nonfunctional alone. When the alpha and omega fragments interact, they become functional and beta-galactosidase protein can be produced. This interaction is known as alpha complementation. It is useful during the screening of beta-galactosidase assay. Artificial galactosides like X-Gal which acts as a substrate for beta-galactosidase and leaves a blue precipitate when hydrolyzed therefore pUC8-transformed E. coli colonies appear blue. They contain plasmids that do not have inserts interrupting the lac Z gene. White colonies consist of bacteria that carry plasmids that have insert-interrupted lac Z genes. (Blue/White Cloning of a DNA Fragment and Assay of β-Galactosidase)

Biochemistry

QUESTION: The Managing Director of a well-known company on Wall Street thrives on a diet of fruit jam, bread, pasta, and coffee. She exercises intermittently. One day she decides to go to her primary healthcare provider for a routine checkup. The healthcare provider recommends that she take the Benedict's test. Assume that the glucose levels of the patient are high. 1. State the results that the test would indicate (specify the color of the solution). 2. State the composition and the properties of the ketohexose derived from fruit jam. 3. Describe the manner in which ketohexose acts as a reducing sugar in the test.

ANSWER: 1. Benedict's test detects reducing sugars (such as glucose). Benedict's reagent contains blue copper ions (Cu2+) which are reduced to copper (Cu+) in the presence of reducing sugars. These are precipitated as red copper(I) oxide which is insoluble in water.Therefore the color of the solution would be Red.

2.Ketohexoses is a ketone containing hexose i.e. it consists of six carbon monosaccharides with a keto group ( R-CO_R ) and other carbon atoms consists an OH group and remaining hydrogens. Ketohexoses are soluble in , doesnt have sharp melting points, can char and sweetin taste because of which are added in the jam. 

3. When the hemi-acetal or ketal hydroxyl group is not engaged or locked or linked to another sugar molecule but  free then the aldehyde (or keto-) form (i.e. the chain-form) is available for reducing copper (II) ions. When a sugar is oxidized, its carbonyl group (i.e. aldehyde or ketone group) is converted to a carboxyl group.
 

Ecogenomics and Its main Objectives

The study of structure and function of a genome in order to understand its relationship between the organism and biotic and abiotic factors of the environment is called ecogenomics. The main objectives of ecogenomics are as follows:

1. To investigate the evolution pattern of an organism with the help of DNA sequences.

2. To discover and explore uncultivable microorganisms with the help of metagenomic techniques.

3. To study the relationship between a species and effect of environmental factors on the species at a molecular level utilizing DNA microarray.

4. To study the effect of genetic and epigenetic variation on evolutionary change due to stress.

5. To study the relationship between different species and gene-environment-function with the help of comparative genomics.

6.  Assessment of region specific biodiversity with the help of DNA microarrays and find the difference between impacted and control regions.

There are several projects going on in order to get more insight into the composition and working of microbes and other soil organisms at a genetic level. For example, a new species of thermophilic bacteria was discovered in order to understand the life sustainable system in the soil by ecogenomics. Samples at a depth of about 8,000 ft below the surface from a gold mine were collected for genome analysis in in South Africa, Witwatersrand Basin. The DNA was extracted from fracture water. Analysis of 16s rDNA in the sample showed that a sulfate reducer and a methylotroph with some thermophilic microbes were present in high quantity. In the thermophiles obtained, there was a dominant Desulfotomaculum like organism discovered which supposedly represented a new species and new family of thermophiles. This organism has several differences from other thermophiles and sulfate reducers in terms of biosustainability in deep subsurface environments so are considered to be a completely new species.

Ecogenomics not only has application in general and pure science but it is also used to understand the function of microbial communities in disease processes which cannot be understood by traditional culture based methods like metagenomics.

While researching the in situ communities like the host system, ecogenomics allows studying the interaction of a pathogen with other microorganisms. This may help to keep a check on the pathogen if the interaction is antagonistic.

With the help of ecogenomics, extensive amounts of genetic information can be analyzed and generated. It can used to explore several factors such as ecological systems which enable the scientists to analyze thoroughly and make more reliable and specific decisions for ecosystem viability.

Ecogenomics can be utilized to determine several aspects altogether as compared to other techniques such as metagenomics. Ecogenomics may investigate factors such as interactions of food web in aquatic environments, spraying effects of fertilizer, ecological effects of different pesticide and land management and functioning in terms of sediment and soil biodiversity. Whereas traditional techniques can analyze limited factors at a time, for example metagenomics studies only the genetics of environmental samples. 

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Genome Sequencing Strategy

Here we have discussed a genome sequencing strategy which is commonly adapted in labs.

The steps in the complete genome sequencing will generate the cDNA sequences that can be used to develop BACs and cDNA microarrays. But since there limited genome data of the organism available, the sequencing will be carried out in two distinct stages.

 

Stage I:

In this stage, the cDNA library of the organism will be generated. The database of novel EST’s from the most diverse EST library would be prepared that can be used for gene annotation, cloning and development of microarray’s etc. The EST’s will expand the understanding of types of genes expressed in the organism . The genome size of the organism will be estimated by comparing the reassociation rate. The genome size will provide a significant information by which we aim to generate BAC genomic DNA library that will inform about GC content of the genome, repetitive sequences and level of polymorphism in the genome.

Stage II:

In this stage, whole genome random shotgun sequencing is proposed for the complete genome sequencing of the organism. This method utilizes comparative genomics protein coding and regulatory regions on the genome. The sequence-tagged BAC clones are mapped to the polytene chromosomes so as assembled scaffolds are directly localized in the genome of the organism  with specific orientations. We aim to produce a draft genome with the knowledge of genomic organization, to carry out the comparative genome analysis and the complete finishing of the genome is done. The finished genome is released to a public domain after getting approval by several committees.